Soybean variety D403301

ABSTRACT

The invention relates to the soybean variety designated D403301. Provided by the invention are the seeds, plants and derivatives of the soybean variety D403301. Also provided by the invention are tissue cultures of the soybean variety D403301 and the plants regenerated therefrom. Still further provided by the invention are methods for producing soybean plants by crossing the soybean variety D403301 with itself or another soybean variety and plants produced by such methods.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of soybeanbreeding. In particular, the invention relates to the novel soybeanvariety D403301.

2. Description of Related Art

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possess the traits to meetthe program goals. The goal is to combine in a single variety animproved combination of desirable traits from the parental germplasm.These important traits may include higher seed yield, resistance todiseases and insects, better stems and roots, tolerance to drought andheat, better agronomic quality, resistance to herbicides, andimprovements in compositional traits.

Soybean, Glycine max (L.), is a valuable field crop. Thus, a continuinggoal of plant breeders is to develop stable, high yielding soybeanvarieties that are agronomically sound. The reasons for this goal are tomaximize the amount of grain produced on the land used and to supplyfood for both animals and humans. To accomplish this goal, the soybeanbreeder must select and develop soybean plants that have the traits thatresult in superior varieties.

The oil extracted from soybeans is widely used in food products, such asmargarine, cooking oil, and salad dressings. Soybean oil is composed ofsaturated, monounsaturated, and polyunsaturated fatty acids, with atypical composition of 11% palmitic, 4% stearic, 25% oleic, 50%linoleic, and 9% linolenic fatty acid content (“Economic Implications ofModified Soybean Traits Summary Report,” Iowa Soybean Promotion Board &American Soybean Association Special Report 92S, May 1990).

SUMMARY OF THE INVENTION

One aspect of the present invention relates to seed of the soybeanvariety D403301. The invention also relates to plants produced bygrowing the seed of the soybean variety D403301, as well as thederivatives of such plants. Further provided are plant parts, includingcells, plant protoplasts, plant cells of a tissue culture from whichsoybean plants can be regenerated, plant calli, plant clumps, and plantcells that are intact in plants or parts of plants, such as pollen,flowers, seeds, pods, leaves, stems, and the like.

In a further aspect, the invention provides a composition comprising aseed of soybean variety D403301 comprised in plant seed growth media. Incertain embodiments, the plant seed growth media is a soil or syntheticcultivation medium. In specific embodiments, the growth medium may becomprised in a container or may, for example, be soil in a field. Plantseed growth media are well known to those of skill in the art andinclude, but are in no way limited to, soil or synthetic cultivationmedium. Advantageously, plant seed growth media can provide adequatephysical support for seeds and can retain moisture and/or nutritionalcomponents. Examples of characteristics for soils that may be desirablein certain embodiments can be found, for instance, in U.S. Pat. Nos.3,932,166 and 4,707,176. Synthetic plant cultivation media are also wellknown in the art and may, in certain embodiments, comprise polymers orhydrogels. Examples of such compositions are described, for example, inU.S. Pat. No. 4,241,537.

Another aspect of the invention relates to a tissue culture ofregenerable cells of the soybean variety D403301, as well as plantsregenerated therefrom, wherein the regenerated soybean plant is capableof expressing all the morphological and physiological characteristics ofa plant grown from the soybean seed designated D403301.

Yet another aspect of the current invention is a soybean plantcomprising a single locus conversion of the soybean variety D403301,wherein the soybean plant is otherwise capable of expressing all themorphological and physiological characteristics of the soybean varietyD403301. In particular embodiments of the invention, the single locusconversion may comprise a transgenic gene which has been introduced bygenetic transformation into the soybean variety D403301 or a progenitorthereof. In still other embodiments of the invention, the single locusconversion may comprise a dominant or recessive allele. The locusconversion may confer potentially any trait upon the single locusconverted plant, including herbicide resistance, insect resistance,resistance to bacterial, fungal, or viral disease, male fertility orsterility, and improved nutritional quality.

Still yet another aspect of the invention relates to a first generation(F₁) hybrid soybean seed produced by crossing a plant of the soybeanvariety D403301 to a second soybean plant. Also included in theinvention are the F₁ hybrid soybean plants grown from the hybrid seedproduced by crossing the soybean variety D403301 to a second soybeanplant. Still further included in the invention are the seeds of an F₁hybrid plant produced with the soybean variety D403301 as one parent,the second generation (F₂) hybrid soybean plant grown from the seed ofthe F₁ hybrid plant, and the seeds of the F₂ hybrid plant.

Still yet another aspect of the invention is a method of producingsoybean seeds comprising crossing a plant of the soybean variety D403301to any second soybean plant, including itself or another plant of thevariety D403301. In particular embodiments of the invention, the methodof crossing comprises the steps of a) planting seeds of the soybeanvariety D403301; b) cultivating soybean plants resulting from said seedsuntil said plants bear flowers; c) allowing fertilization of the flowersof said plants; and d) harvesting seeds produced from said plants.

Still yet another aspect of the invention is a method of producinghybrid soybean seeds comprising crossing the soybean variety D403301 toa second, distinct soybean plant which is nonisogenic to the soybeanvariety D403301. In particular embodiments of the invention, thecrossing comprises the steps of a) planting seeds of soybean varietyD403301 and a second, distinct soybean plant, b) cultivating the soybeanplants grown from the seeds until the plants bear flowers; c) crosspollinating a flower on one of the two plants with the pollen of theother plant, and d) harvesting the seeds resulting from the crosspollinating.

Still yet another aspect of the invention is a method for developing asoybean plant in a soybean breeding program comprising: obtaining asoybean plant, or its parts, of the variety D403301; and b) employingsaid plant or parts as a source of breeding material using plantbreeding techniques. In the method, the plant breeding techniques may beselected from the group consisting of recurrent selection, massselection, bulk selection, backcrossing, pedigree breeding, geneticmarker-assisted selection and genetic transformation. In certainembodiments of the invention, the soybean plant of variety D403301 isused as the male or female parent.

Still yet another aspect of the invention is a method of producing asoybean plant derived from the soybean variety D403301, the methodcomprising the steps of: (a) preparing a progeny plant derived fromsoybean variety D403301 by crossing a plant of the soybean varietyD403301 with a second soybean plant; and (b) crossing the progeny plantwith itself or a second plant to produce a progeny plant of a subsequentgeneration which is derived from a plant of the soybean variety D403301.In one embodiment of the invention, the method further comprises: (c)crossing the progeny plant of a subsequent generation with itself or asecond plant; and (d) repeating steps (b) and (c) for, in someembodiments, at least 2, 3, 4 or more additional generations to producean inbred soybean plant derived from the soybean variety D403301. Alsoprovided by the invention is a plant produced by this and the othermethods of the invention.

In another embodiment of the invention, the method of producing asoybean plant derived from the soybean variety D403301 furthercomprises: (a) crossing the soybean variety D403301-derived soybeanplant with itself or another soybean plant to yield additional soybeanvariety D403301-derived progeny soybean seed; (b) growing the progenysoybean seed of step (a) under plant growth conditions to yieldadditional soybean variety D403301-derived soybean plants; and (c)repeating the crossing and growing steps of (a) and (b) to generatefurther soybean variety D403301-derived soybean plants. In specificembodiments, steps (a) and (b) may be repeated at least 1, 2, 3, 4, or 5or more times as desired. The invention still further provides a soybeanplant produced by this and the foregoing methods.

DETAILED DESCRIPTION OF THE INVENTION

The instant invention provides methods and composition relating toplants, seeds and derivatives of the soybean variety D403301. Soybeanvariety D403301 is adapted to early group III. Soybean variety D403301was developed from an initial cross of BL3309D1-G0RN/(AG3430). Thebreeding history of the variety can be summarized as follows:

Generation Year Description Cross 2009 The cross was made near Findlay,OH. F₁ 2009 Plants were grown near Kunia, HI and advanced using singleplant selection. F₂ 2010 Plants were grown near Kunia, HI and advancedusing bulk. F₃ 2010 Plants were grown near Findlay, OH and advancedusing single plant selection. F₄ 2010 Plants were grown near Rancagua,Chile in Progeny Rows and the variety D403301 was selected based on theagronomic characteristics, including but not limited to, general planthealth, lodging, early emergence, and general disease resistance,including PRR, SCN, etc. Yield Testing Generation Year No. of LocationsRank No. of Entries F₅ 2011 6 3 80 F₆ 2012 38 13 60 F₇ 2013 34 12 40

The soybean variety D403301 has been judged to be uniform for breedingpurposes and testing. The variety D403301 can be reproduced by plantingand growing seeds of the variety under self-pollinating orsib-pollinating conditions, as is known to those of skill in theagricultural arts. Variety D403301 shows no variants other than whatwould normally be expected due to environment or that would occur foralmost any characteristic during the course of repeated sexualreproduction.

The results of an objective evaluation of the variety are presentedbelow, in Table 1. Those of skill in the art will recognize that theseare typical values that may vary due to environment and that othervalues that are substantially equivalent are within the scope of theinvention. An ‘*’ denotes classifications/scores generated based ongreenhouse assays.

TABLE 1 Phenotypic Description of Variety D403301 Trait PhenotypeMorphology: Relative Maturity 3.3 Flower Color Purple Pubescence ColorGray Hilum Color Imperfect Black Pod Color Brown Seed Coat Color YellowSeed Coat Luster Dull Seed Shape Spherical flattened Cotyledon ColorYellow Leaf Shape Ovate Leaf Color Green Canopy Intermediate GrowthHabit Indeterminate Disease Reactions Phytophthora Allele* Segregatingfor Rps1c and Rps1k Phytophthora Tolerance* (Race 25) SusceptibleSoybean Cyst Nematode Race 3* Resistant Soybean Cyst Nematode Race 1*Susceptible Brown Stem Rot* Moderately resistant Sudden Death Syndrome*Moderately resistant Chloride Sensitivity* Includer Herbicide Reactions:Glyphosate Resistant, MON89788 Sulfonylurea Susceptible Fatty acid:Fatty acid composition Normal

As disclosed herein above, soybean variety D403301 contains eventMON89788. Event MON89788, also known as event GM_A19788, confersglyphosate tolerance and is the subject of U.S. Pat. No. 7,632,985, thedisclosure of which is incorporated herein by reference. Event MON89788is also covered by one or more of the following patents: U.S. Pat. Nos.8,053,184; 7,608,761; 7,141,722; 6,949,696; 6,660,911; 6,051,753;5,728,925; 5,717,084.

The performance characteristics of soybean variety D403301 were alsoanalyzed and comparisons were made with selected varieties. The resultsof the analysis are presented below, in Table 2.

TABLE 2 Exemplary Agronomic Traits of Variety D403301 and SelectedVarieties Entries Compared YLD_BE MAT PHT LDG EMR SDV PRO OIL SWT IDCD403301 58.4 26 40.9 3.3 1.9 3.7 39.4 21.1 2,230.3 1 AG3231 56.3 25.839.5 1.5 2.1 3.9 39.9 20.6 2,283 1 Deviation 2.08 0.17 1.37 1.78 −0.18−0.23 −0.52 0.54 −52.67 0 Significance ** + ** * ** # Obs 102 20 13 3222 17 4 4 3 1 Years 3 3 3 3 3 2 1 1 1 1 Win Percent 67 53 15 3 57 64 0100 33 — Test Mean 56 24.6 40 2.5 2.1 3.8 40.4 20.9 2,388 1 D403301 5625.2 41.3 3.2 2.2 3 39.3 21.1 2,230.3 AG3333 54.3 27 38 2.2 2.4 3.3 40.620.9 1,897.3 Deviation 1.64 −1.81 3.33 0.97 −0.24 −0.31 −1.24 0.2 333Significance * * ** # Obs 56 10 8 9 13 4 4 4 3 Years 3 3 2 3 3 2 1 1 1Win Percent 66 70 12 38 73 67 25 75 100 Test Mean 55.1 26.1 40.5 2.5 2.33.1 40.5 20.7 2,367.8 D403301 57.5 25.3 40.2 3.1 1.9 3.6 1 AG3334 5825.6 41.1 1.5 1.8 3.8 1 Deviation −0.58 −0.38 −0.95 1.62 0.08 −0.22 0Significance ** # Obs 88 15 10 21 20 15 1 Years 3 3 3 3 3 2 1 WinPercent 50 67 60 0 43 80 — Test Mean 55.8 25.4 39.5 2.3 2 3.7 1 D40330156.8 26.2 40.9 2.9 1.9 2.6 39.3 21.1 2,230.3 AG3431 56.1 28.5 40.2 1.72.1 3.1 40.4 20.7 2,469.3 Deviation 0.63 −2.35 0.64 1.14 −0.23 −0.52−1.05 0.48 −239 Significance ** ** * + * * # Obs 80 16 13 16 22 5 4 4 3Years 3 3 3 3 3 2 1 1 1 Win Percent 59 88 23 20 75 100 0 100 0 Test Mean54.6 26.4 40.2 2.2 2.1 2.9 40.6 20.7 2,348.1 D403301 57.6 25.8 40.9 2.91.9 3.6 39.4 21.1 2,230.3 1 AG3432 58.5 28.1 41.7 3.1 2 4 40.1 212,210.7 1 Deviation −0.86 −2.3 −0.78 −0.17 −0.13 −0.41 −0.79 0.16 19.670 Significance + ** + # Obs 99 21 13 29 22 19 4 4 3 1 Years 3 3 3 3 3 21 1 1 1 Win Percent 43 90 69 57 68 77 25 50 67 — Test Mean 56.1 26.840.3 2.3 2.1 3.8 40.6 20.7 2,348.1 1 D403301 53.8 26.6 41.8 3.2 2 3.239.4 21.1 2,230.3 CBRB3201R2N 52 22.6 40.7 1.8 2.5 3.3 40.4 20.9 2,253.2Deviation 1.81 3.99 1.1 1.33 −0.42 −0.17 −1.07 0.22 −22.83Significance * * + ** # Obs 42 7 8 9 12 3 4 4 3 Years 2 2 2 2 2 1 1 1 1Win Percent 67 57 25 12 75 100 0 75 33 Test Mean 52.4 24.3 40.8 2.5 2.23.2 40.4 20.8 2,366.7 D403301 57.3 25.7 40.9 3 1.9 3.6 39.4 21.1 2,230.31 CBRB3391R2N 56.4 24.9 40.3 2.1 2.1 4 40.2 20.5 2,398.7 1 Deviation0.99 0.86 0.56 0.85 −0.2 −0.39 −0.81 0.67 −168.33 0 Significance +** * + * # Obs 96 19 11 28 21 19 4 4 3 1 Years 3 3 3 3 3 2 1 1 1 1 WinPercent 59 63 36 17 65 93 0 100 33 — Test Mean 56.2 25.7 40.4 2.3 2 3.840.5 20.7 2,367.8 1 D403301 57.6 24.7 40 2.9 1.9 3.7 39.4 21.1 2,230.3 1CR2 3422N 57.9 25.4 40.5 1.6 2 3.6 40.8 20.7 2,110 1 Deviation −0.36−0.71 −0.49 1.28 −0.05 0.05 −1.4 0.4 120.33 0 Significance ** + + # Obs100 18 11 28 21 20 4 4 3 1 Years 3 3 3 3 3 2 1 1 1 1 Win Percent 50 8170 8 38 62 0 100 67 — Test Mean 56.9 26.1 39.9 2.4 2 3.8 40.5 20.72,367.8 1 D403301 58.4 25.7 40.5 3.3 1.9 3.6 39.4 21.1 2,230.3 1 CR3222N57.8 26.7 39.2 2.8 2.4 3.7 39.5 21.7 2,190.3 1 Deviation 0.56 −1.02 1.290.55 −0.43 −0.14 −0.11 −0.53 40 0 Significance + ** ** # Obs 96 18 11 3021 18 4 4 3 1 Years 3 3 3 3 3 2 1 1 1 1 Win Percent 54 82 30 26 78 64 500 67 — Test Mean 57.2 25.8 39.8 2.5 2 3.7 40.5 20.7 2,367.8 1 D40330157.4 25.2 40.7 3.1 1.9 3.6 1 CR3302N 58.4 27.9 39.8 1.9 1.9 3.9 1Deviation −0.93 −2.71 0.81 1.21 0 −0.35 0 Significance ** ** + # Obs 8715 10 21 20 15 1 Years 3 3 3 3 3 2 1 Win Percent 44 87 33 11 47 75 —Test Mean 55.9 25.5 40.1 2.3 2 3.7 1 D403301 57.5 25.5 40 3 1.9 3.6 39.421.1 2,230.3 1 CR3333N 57.5 26.2 39.3 2.9 1.9 3.7 39.7 21.4 2,556.3 1Deviation −0.03 −0.7 0.77 0.08 0.04 −0.08 −0.31 −0.24 −326 0Significance * # Obs 97 19 11 27 21 19 4 4 3 1 Years 3 3 3 3 3 2 1 1 1 1Win Percent 49 69 12 45 60 75 50 50 0 — Test Mean 56.5 26.3 39.7 2.3 2.13.8 40.5 20.7 2,367.8 1 D403301 57.3 25.3 40.7 3 1.9 3.6 1 CR3462N 58.328.3 41.1 2.5 2.1 3.7 1 Deviation −1.06 −2.98 −0.41 0.48 −0.13 −0.06 0Significance ** + # Obs 94 17 10 26 20 18 1 Years 3 3 3 3 3 2 1 WinPercent 44 100 44 20 58 70 — Test Mean 56.3 26.4 39.9 2.3 2 3.8 1D403301 56.7 26.2 40.4 2.5 1.8 2.5 93Y40 53 25.6 38.9 1.5 2.3 2.6Deviation 3.69 0.54 1.57 0.93 −0.46 −0.12 Significance ** ** * # Obs 6310 7 10 17 4 Years 3 3 3 3 3 2 Win Percent 70 80 29 0 83 50 Test Mean55.8 26.3 40.2 1.9 1.9 2.8 D403301 60.1 25.3 38.3 2.9 1.8 3.7 1 93Y4356.7 25.4 38.3 2.1 2 3.8 1 Deviation 3.42 −0.07 0 0.74 −0.22 −0.16 0Significance ** ** # Obs 54 11 3 19 9 16 1 Years 1 1 1 1 1 1 1 WinPercent 70 43 67 21 60 73 — Test Mean 59.2 26.4 38.2 2.2 1.8 3.9 1 **,*, + Significant at P < 0.01, 0.05, or 0.10, respectivelyI. Breeding Soybean Variety D403301

One aspect of the current invention concerns methods for crossing thesoybean variety D403301 with itself or a second plant and the seeds andplants produced by such methods. These methods can be used forpropagation of the soybean variety D403301, or can be used to producehybrid soybean seeds and the plants grown therefrom. Hybrid soybeanplants can be used by farmers in the commercial production of soyproducts or may be advanced in certain breeding protocols for theproduction of novel soybean varieties. A hybrid plant can also be usedas a recurrent parent at any given stage in a backcrossing protocolduring the production of a single locus conversion of the soybeanvariety D403301.

Soybean variety D403301 is well suited to the development of newvarieties based on the elite nature of the genetic background of thevariety. In selecting a second plant to cross with D403301 for thepurpose of developing novel soybean varieties, it will typically bedesired to choose those plants which either themselves exhibit one ormore selected desirable characteristics or which exhibit the desiredcharacteristic(s) when in hybrid combination. Examples of potentiallydesired characteristics include seed yield, lodging resistance,emergence, seedling vigor, disease tolerance, maturity, plant height,high oil content, high protein content and shattering resistance.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of variety used commercially (e.g., F₁ hybrid variety, purelinevariety, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, recurrent selection andbackcrossing.

The complexity of inheritance influences choice of the breeding method.Backcross breeding is used to transfer one or a few favorable genes fora highly heritable trait into a desirable variety. This approach hasbeen used extensively for breeding disease-resistant varieties (Bowerset al., Crop Sci., 32(1):67-72, 1992; Nickell and Bernard, Crop Sci.,32(3):835, 1992). Various recurrent selection techniques are used toimprove quantitatively inherited traits controlled by numerous genes.The use of recurrent selection in self-pollinating crops depends on theease of pollination, the frequency of successful hybrids from eachpollination, and the number of hybrid offspring from each successfulcross.

Each breeding program should include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard,overall value of the advanced breeding lines, and number of successfulvarieties produced per unit of input (e.g., per year, per dollarexpended, etc.).

Promising advanced breeding lines are thoroughly tested and compared toappropriate standards in environments representative of the commercialtarget area(s) for generally three or more years. The best lines arecandidates for new commercial varieties. Those still deficient in a fewtraits may be used as parents to produce new populations for furtherselection.

These processes, which lead to the final step of marketing anddistribution, may take as much as eight to 12 years from the time thefirst cross is made. Therefore, development of new varieties is atime-consuming process that requires precise forward planning, efficientuse of resources, and a minimum of changes in direction.

A most difficult task is the identification of individuals that aregenetically superior, because for most traits, the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to one or more widely grownstandard varieties. Single observations are generally inconclusive,while replicated observations provide a better estimate of geneticworth.

The goal of plant breeding is to develop new, unique and superiorsoybean varieties and hybrids. The breeder initially selects and crossestwo or more parental lines, followed by repeated selfing and selection,producing many new genetic combinations. Each year, the plant breederselects the germplasm to advance to the next generation. This germplasmis grown under unique and different geographical, climatic and soilconditions, and further selections are then made, during and at the endof the growing season. The varieties which are developed areunpredictable. This unpredictability is because the breeder's selectionoccurs in unique environments, with no control at the DNA level (usingconventional breeding procedures), and with millions of differentpossible genetic combinations being generated. A breeder of ordinaryskill in the art cannot predict the final resulting lines he develops,except possibly in a very gross and general fashion. The same breedercannot produce the same variety twice by using the exact same originalparents and the same selection techniques. This unpredictability resultsin the expenditure of large amounts of research monies to developsuperior new soybean varieties.

Pedigree breeding and recurrent selection breeding methods are used todevelop varieties from breeding populations. Breeding programs combinedesirable traits from two or more varieties or various broad-basedsources into breeding pools from which varieties are developed byselfing and selection of desired phenotypes. The new varieties areevaluated to determine which have commercial potential.

Pedigree breeding is commonly used for the improvement ofself-pollinating crops. Two parents which possess favorable,complementary traits are crossed to produce an F₁. An F₂ population isproduced by selfing one or several F₁'s. Selection of the bestindividuals may begin in the F₂ population (or later depending upon thebreeder's objectives); then, beginning in the F₃, the best individualsin the best families can be selected. Replicated testing of families canbegin in the F₃ or F₄ generation to improve the effectiveness ofselection for traits with low heritability. At an advanced stage ofinbreeding (i.e., F₆ and F₇), the best lines or mixtures ofphenotypically similar lines are tested for potential release as newvarieties.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

Backcross breeding has been used to transfer genetic loci for simplyinherited, highly heritable traits into a desirable homozygous varietywhich is the recurrent parent. The source of the trait to be transferredis called the donor or nonrecurent parent. The resulting plant isexpected to have the attributes of the recurrent parent (i.e., variety)and the desirable trait transferred from the donor parent. After theinitial cross, individuals possessing the phenotype of the donor parentare selected and repeatedly crossed (backcrossed) to the recurrentparent. The resulting plant is expected to have the attributes of therecurrent parent (i.e., variety) and the desirable trait transferredfrom the donor parent.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F₂ plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

In a multiple-seed procedure, soybean breeders commonly harvest one ormore pods from each plant in a population and thresh them together toform a bulk. Part of the bulk is used to plant the next generation andpart is put in reserve. This procedure is also referred to as modifiedsingle-seed descent or the pod-bulk technique.

The multiple-seed procedure has been used to save labor at harvest. Itis considerably faster to thresh pods with a machine than to remove oneseed from each by hand for the single-seed procedure. The multiple-seedprocedure also makes it possible to plant the same number of seeds of apopulation each generation of inbreeding. Enough seeds are harvested tomake up for those plants that did not germinate or produce seed.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., Allard, “Principles of plant breeding,” John Wiley & Sons,NY, University of California, Davis, Calif., 50-98, 1960; Simmonds,“Principles of crop improvement,” Longman, Inc., NY, 369-399, 1979;Sneep and Hendriksen, “Plant breeding perspectives,” Wageningen (ed),Center for Agricultural Publishing and Documentation, 1979; Fehr, In:Soybeans: Improvement, Production and Uses,” 2d Ed., Manograph 16:249,1987; Fehr, “Principles of cultivar development,” Theory and Technique(Vol 1) and Crop Species Soybean (Vol 2), Iowa State Univ., MacmillianPub. Co., NY, 360-376, 1987; Poehlman and Sleper, “Breeding Field Crops”Iowa State University Press, Ames, 1995; Sprague and Dudley, eds., Cornand Improvement, 5th ed., 2006).

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current varieties. In addition toshowing superior performance, there must be a demand for a new varietythat is compatible with industry standards or which creates a newmarket. The introduction of a new variety will incur additional costs tothe seed producer, the grower, processor and consumer; for specialadvertising and marketing, altered seed and commercial productionpractices, and new product utilization. The testing preceding release ofa new variety should take into consideration research and developmentcosts as well as technical superiority of the final variety. Forseed-propagated varieties, it must be feasible to produce seed easilyand economically.

In addition to phenotypic observations, a plant can also be identifiedby its genotype. The genotype of a plant can be characterized through amolecular marker profile, which can identify plants of the same varietyor a related variety, can identify plants and plant parts which aregenetically superior as a result of an event comprising a backcrossconversion, transgene, or genetic sterility factor, or can be used todetermine or validate a pedigree. Such molecular marker profiling can beaccomplished using a variety of techniques including, but not limitedto, restriction fragment length polymorphism (RFLP), amplified fragmentlength polymorphism (AFLP), sequence-tagged sites (STS), randomlyamplified polymorphic DNA (RAPD), arbitrarily primed polymerase chainreaction (AP-PCR), DNA amplification fingerprinting (DAF), sequencecharacterized amplified regions (SCARs), variable number tandem repeat(VNTR), short tandem repeat (STR), single feature polymorphism (SFP),simple sequence length polymorphism (SSLP), restriction site associatedDNA, allozymes, isozyme markers, single nucleotide polymorphisms (SNPs),or simple sequence repeat (SSR) markers, also known as microsatellites(Gupta et al., 1999; Korzun et al., 2001). Various types of thesemarkers, for example, can be used to identify individual varietiesdeveloped from specific parent varieties, as well as cells or otherplant parts thereof. For example, see Cregan et al. (1999) “AnIntegrated Genetic Linkage Map of the Soybean Genome” Crop Science39:1464-1490, and Berry et al. (2003) “Assessing Probability of AncestryUsing Simple Sequence Repeat Profiles: Applications to Maize InbredLines and Soybean Varieties” Genetics 165:331-342, each of which areincorporated by reference herein in their entirety.

In some examples, one or more markers may be used to characterize and/orevaluate a soybean variety. Particular markers used for these purposesare not limited to any particular set of markers, but are envisioned toinclude any type of marker and marker profile that provides a means fordistinguishing varieties. One method of comparison may to use onlyhomozygous loci for soybean variety D403301.

Primers and PCR protocols for assaying these and other markers aredisclosed in, for example, Soybase (sponsored by the USDA AgriculturalResearch Service and Iowa State University) located on the world wideweb at 129.186.26/94/SSR.html. In addition to being used foridentification of soybean variety D403301, as well as plant parts andplant cells of soybean variety D403301, a genetic profile may be used toidentify a soybean plant produced through the use of soybean varietyD403301 or to verify a pedigree for progeny plants produced through theuse of soybean variety D403301. A genetic marker profile may also beuseful in breeding and developing backcross conversions.

In an embodiment, the present invention provides a soybean plantcharacterized by molecular and physiological data obtained from arepresentative sample of said variety deposited with the American TypeCulture Collection (ATCC). Thus, plants, seeds, or parts thereof, havingall or essentially all of the morphological and physiologicalcharacteristics of soybean variety D403301 are provided. Furtherprovided is a soybean plant formed by the combination of the disclosedsoybean plant or plant cell with another soybean plant or cell andcomprising the homozygous alleles of the variety.

In some examples, a plant, a plant part, or a seed of soybean varietyD403301 may be characterized by producing a molecular profile. Amolecular profile may include, but is not limited to, one or moregenotypic and/or phenotypic profile(s). A genotypic profile may include,but is not limited to, a marker profile, such as a genetic map, alinkage map, a trait maker profile, a SNP profile, an SSR profile, agenome-wide marker profile, a haplotype, and the like. A molecularprofile may also be a nucleic acid sequence profile, and/or a physicalmap. A phenotypic profile may include, but is not limited to, a proteinexpression profile, a metabolic profile, an mRNA expression profile, andthe like.

One means of performing genetic marker profiles is using SSRpolymorphisms that are well known in the art. A marker system based onSSRs can be highly informative in linkage analysis relative to othermarker systems, in that multiple alleles may be present. Anotheradvantage of this type of marker is that through use of flankingprimers, detection of SSRs can be achieved, for example, by using thepolymerase chain reaction (PCR), thereby eliminating the need forlabor-intensive Southern hybridization. PCR detection may be performedusing two oligonucleotide primers flanking the polymorphic segment ofrepetitive DNA to amplify the SSR region.

Following amplification, markers can be scored by electrophoresis of theamplification products. Scoring of marker genotype is based on the sizeof the amplified fragment, which correlates to the number of base pairsof the fragment. While variation in the primer used or in the laboratoryprocedures can affect the reported fragment size, relative values shouldremain constant regardless of specific primer or laboratory used. Whencomparing varieties, it may be beneficial to have all profiles performedin the same lab. Primers that can be used are publically available andmay be found in, for example, Soybase or Cregan (Crop Science39:1464-1490, 1999).

A genotypic profile of soybean variety D403301 can be used to identify aplant comprising variety D403301 as a parent, since such plants willcomprise the same homozygous alleles as variety D403301. Because thesoybean variety is essentially homozygous at all relevant loci, mostloci should have only one type of allele present. In contrast, a geneticmarker profile of an F1 progeny should be the sum of those parents,e.g., if one parent was homozygous for allele X at a particular locus,and the other parent homozygous for allele Y at that locus, then the F1progeny will be XY (heterozygous) at that locus. Subsequent generationsof progeny produced by selection and breeding are expected to be ofgenotype XX (homozygous), YY (homozygous), or XY (heterozygous) for thatlocus position. When the F1 plant is selfed or sibbed for successivefilial generations, the locus should be either X or Y for that position.

In addition, plants and plant parts substantially benefiting from theuse of variety D403301 in their development, such as variety D403301comprising a backcross conversion, transgene, or genetic sterilityfactor, may be identified by having a molecular marker profile with ahigh percent identity to soybean variety D403301. Such a percentidentity might be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.5% or 99.9% identical to soybean variety D403301.

A genotypic profile of variety D403301 also can be used to identifyessentially derived varieties and other progeny varieties developed fromthe use of variety D403301, as well as cells and other plant partsthereof. Plants of the invention include any plant having at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of themarkers in the genotypic profile, and that retain 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of the morphological andphysiological characteristics of variety D403301 when grown under thesame conditions. Such plants may be developed using markers well knownin the art. Progeny plants and plant parts produced using varietyD403301 may be identified, for example, by having a molecular markerprofile of at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% geneticcontribution from soybean variety D403301, as measured by either percentidentity or percent similarity. Such progeny may be furthercharacterized as being within a pedigree distance of variety D403301,such as within 1, 2, 3, 4, or 5 or less cross pollinations to a soybeanplant other than variety D403301, or a plant that has variety D403301 asa progenitor. Unique molecular profiles may be identified with othermolecular tools, such as SNPs and RFLPs.

Any time the soybean variety D403301 is crossed with another, different,variety, first generation (F₁) soybean progeny are produced. The hybridprogeny are produced regardless of characteristics of the two varietiesproduced. As such, an F₁ hybrid soybean plant may be produced bycrossing D403301 with any second soybean plant. The second soybean plantmay be genetically homogeneous (e.g., inbred) or may itself be a hybrid.Therefore, any F₁ hybrid soybean plant produced by crossing soybeanvariety D403301 with a second soybean plant is a part of the presentinvention.

Soybean plants (Glycine max L.) can be crossed by either natural ormechanical techniques (see, e.g., Fehr, “Soybean,” In: Hybridization ofCrop Plants, Fehr and Hadley (eds), Am. Soc. Agron. and Crop Sci. Soc.Am., Madison, Wis., 590-599, 1980). Natural pollination occurs insoybeans either by self pollination or natural cross pollination, whichtypically is aided by pollinating organisms. In either natural orartificial crosses, flowering and flowering time are an importantconsideration. Soybean is a short-day plant, but there is considerablegenetic variation for sensitivity to photoperiod (Hamner, “Glycinemax(L.) Merrill,” In: The Induction of Flowering: Some Case Histories,Evans (ed), Cornell Univ. Press, Ithaca, N.Y., 62-89, 1969; Criswell andHume, Crop Sci., 12:657-660, 1972). The critical day length forflowering ranges from about 13 h for genotypes adapted to tropicallatitudes to 24 h for photoperiod-insensitive genotypes grown at higherlatitudes (Shibles et al., “Soybean,” In: Crop Physiology, Some CaseHistories, Evans (ed), Cambridge Univ. Press, Cambridge, England,51-189, 1975). Soybeans seem to be insensitive to day length for 9 daysafter emergence. Photoperiods shorter than the critical day length arerequired for 7 to 26 days to complete flower induction (Borthwick andParker, Bot. Gaz., 100:374-387, 1938; Shanmugasundaram and Tsou, CropSci., 18:598-601, 1978).

Sensitivity to day length is an important consideration when genotypesare grown outside of their area of adaptation. When genotypes adapted totropical latitudes are grown in the field at higher latitudes, they maynot mature before frost occurs. Plants can be induced to flower andmature earlier by creating artificially short days or by grafting (Fehr,“Soybean,” In: Hybridization of Crop Plants, Fehr and Hadley (eds), Am.Soc. Agron. and Crop Sci. Soc. Am., Madison, Wis., 590-599, 1980).Soybeans frequently are grown in winter nurseries located at sea levelin tropical latitudes where day lengths are much shorter than theircritical photoperiod. The short day lengths and warm temperaturesencourage early flowering and seed maturation, and genotypes can producea seed crop in 90 days or fewer after planting. Early flowering isuseful for generation advance when only a few self-pollinated seeds perplant are needed, but not for artificial hybridization because theflowers self-pollinate before they are large enough to manipulate forhybridization. Artificial lighting can be used to extend the natural daylength to about 14.5 h to obtain flowers suitable for hybridization andto increase yields of self-pollinated seed.

The effect of a short photoperiod on flowering and seed yield can bepartly offset by altitude, probably due to the effects of cooltemperature (Major et al., Crop Sci., 15:174-179, 1975). At tropicallatitudes, varieties adapted to the northern U.S. perform more likethose adapted to the southern U.S. at high altitudes than they do at sealevel.

The light level required to delay flowering is dependent on the qualityof light emitted from the source and the genotype being grown. Bluelight with a wavelength of about 480 nm requires more than 30 times theenergy to inhibit flowering as red light with a wavelength of about 640nm (Parker et al., Bot. Gaz., 108:1-26, 1946).

Temperature can also play a significant role in the flowering anddevelopment of soybean (Major et al., Crop Sci., 15:174-179, 1975). Itcan influence the time of flowering and suitability of flowers forhybridization. Temperatures below 21° C. or above 32° C. can reducefloral initiation or seed set (Hamner, “Glycine max(L.) Merrill,” In:The Induction of Flowering: Some Case Histories, Evans (ed), CornellUniv. Press, Ithaca, N.Y., 62-89, 1969; van Schaik and Probst, Agron.J., 50:192-197, 1958). Artificial hybridization is most successfulbetween 26° C. and 32° C. because cooler temperatures reduce pollen shedand result in flowers that self-pollinate before they are large enoughto manipulate. Warmer temperatures frequently are associated withincreased flower abortion caused by moisture stress; however, successfulcrosses are possible at about 35° C. if soil moisture is adequate.

Soybeans have been classified as indeterminate, semi-determinate, anddeterminate based on the abruptness of stem termination after floweringbegins (Bernard and Weiss, “Qualitative genetics,” In: Soybeans:Improvement, Production, and Uses, Caldwell (ed), Am. Soc. of Agron.,Madison, Wis., 117-154, 1973). When grown at their latitude ofadaptation, indeterminate genotypes flower when about one-half of thenodes on the main stem have developed. They have short racemes with fewflowers, and their terminal node has only a few flowers.Semi-determinate genotypes also flower when about one-half of the nodeson the main stem have developed, but node development and flowering onthe main stem stops more abruptly than on indeterminate genotypes. Theirracemes are short and have few flowers, except for the terminal one,which may have several times more flowers than those lower on the plant.Determinate varieties begin flowering when all or most of the nodes onthe main stem have developed. They usually have elongated racemes thatmay be several centimeters in length and may have a large number offlowers. Stem termination and flowering habit are reported to becontrolled by two major genes (Bernard and Weiss, “Qualitativegenetics,” In: Soybeans: Improvement, Production, and Uses, Caldwell(ed), Am. Soc. of Agron., Madison, Wis., 117-154, 1973).

Soybean flowers typically are self-pollinated on the day the corollaopens. The amount of natural crossing, which is typically associatedwith insect vectors such as honeybees, is approximately 1% for adjacentplants within a row and 0.5% between plants in adjacent rows (Boerma andMoradshahi, Crop Sci., 15:858-861, 1975). The structure of soybeanflowers is similar to that of other legume species and consists of acalyx with five sepals, a corolla with five petals, 10 stamens, and apistil (Carlson, “Morphology”, In: Soybeans: Improvement, Production,and Uses, Caldwell (ed), Am. Soc. of Agron., Madison, Wis., 17-95,1973). The calyx encloses the corolla until the day before anthesis. Thecorolla emerges and unfolds to expose a standard, two wing petals, andtwo keel petals. An open flower is about 7 mm long from the base of thecalyx to the tip of the standard and 6 mm wide across the standard. Thepistil consists of a single ovary that contains one to five ovules, astyle that curves toward the standard, and a club-shaped stigma. Thestigma is receptive to pollen about 1 day before anthesis and remainsreceptive for 2 days after anthesis, if the flower petals are notremoved. Filaments of nine stamens are fused, and the one nearest thestandard is free. The stamens form a ring below the stigma until about 1day before anthesis, then their filaments begin to elongate rapidly andelevate the anthers around the stigma. The anthers dehisce on the day ofanthesis, pollen grains fall on the stigma, and within 10 h the pollentubes reach the ovary and fertilization is completed (Johnson andBernard, “Soybean genetics and breeding,” In: The Soybean, Norman (ed),Academic Press, NY, 1-73, 1963).

Self-pollination occurs naturally in soybean with no manipulation of theflowers. For the crossing of two soybean plants, it is often beneficial,although not required, to utilize artificial hybridization. Inartificial hybridization, the flower used as a female in a cross ismanually cross pollinated prior to maturation of pollen from the flower,thereby preventing self fertilization, or alternatively, the male partsof the flower are emasculated using a technique known in the art.Techniques for emasculating the male parts of a soybean flower include,for example, physical removal of the male parts, use of a genetic factorconferring male sterility, and application of a chemical gametocide tothe male parts.

For artificial hybridization employing emasculation, flowers that areexpected to open the following day are selected on the female parent.The buds are swollen and the corolla is just visible through the calyxor has begun to emerge. The selected buds on a parent plant areprepared, and all self-pollinated flowers or immature buds are removed.Special care is required to remove immature buds that are hidden underthe stipules at the leaf axil, and which could develop into flowers at alater date. To remove flowers, the flower is grasped and the location ofthe stigma determined by examining the sepals. A long, curvy sepalcovers the keel, and the stigma is on the opposite side of the flower.The calyx is removed by pulling each sepal down and around the flower,and the exposed corolla is removed just above the calyx scar, takingcare to remove the keel petals without injuring the stigma. The ring ofanthers is visible after the corolla is removed, unless the anthers wereremoved with the petals. Cross-pollination can then be carried outusing, for example, petri dishes or envelopes in which male flowers havebeen collected. Desiccators containing calcium chloride crystals areused in some environments to dry male flowers to obtain adequate pollenshed.

It has been demonstrated that emasculation is unnecessary to preventself-pollination (Walker et al., Crop Sci., 19:285-286, 1979). Whenemasculation is not used, the anthers near the stigma frequently areremoved to make it clearly visible for pollination. The female flowerusually is hand-pollinated immediately after it is prepared; although adelay of several hours does not seem to reduce seed set. Pollen shedtypically begins in the morning and may end when temperatures are above30° C., or may begin later and continue throughout much of the day withmore moderate temperatures.

Pollen is available from a flower with a recently opened corolla, butthe degree of corolla opening associated with pollen shed may varyduring the day. In many environments, it is possible to collect maleflowers and use them immediately without storage. In the southern U.S.and other humid climates, pollen shed occurs in the morning when femaleflowers are more immature and difficult to manipulate than in theafternoon, and the flowers may be damp from heavy dew. In thosecircumstances, male flowers may be collected into envelopes or petridishes in the morning and the open container placed in a desiccator forabout 4 h at a temperature of about 25° C. The desiccator may be takento the field in the afternoon and kept in the shade to prevent excessivetemperatures from developing within it. Pollen viability can bemaintained in flowers for up to 2 days when stored at about 5° C. In adesiccator at 3° C., flowers can be stored successfully for severalweeks; however, varieties may differ in the percentage of pollen thatgerminates after long-term storage (Kuehl, “Pollen viability and stigmareceptivity of Glycine max (L.) Merrill,” Thesis, North Carolina StateCollege, Raleigh, N.C., 1961).

Either with or without emasculation of the female flower, handpollination can be carried out by removing the stamens and pistil with aforceps from a flower of the male parent and gently brushing the anthersagainst the stigma of the female flower. Access to the stamens can beachieved by removing the front sepal and keel petals, or piercing thekeel with closed forceps and allowing them to open to push the petalsaway. Brushing the anthers on the stigma causes them to rupture, and thehighest percentage of successful crosses is obtained when pollen isclearly visible on the stigma. Pollen shed can be checked by tapping theanthers before brushing the stigma. Several male flowers may have to beused to obtain suitable pollen shed when conditions are unfavorable, orthe same male may be used to pollinate several flowers with good pollenshed.

When male flowers do not have to be collected and dried in a desiccator,it may be desired to plant the parents of a cross adjacent to eachother. Plants usually are grown in rows 65 to 100 cm apart to facilitatemovement of personnel within the field nursery. Yield of self-pollinatedseed from an individual plant may range from a few seeds to more than1,000 as a function of plant density. A density of 30 plants/m of rowcan be used when 30 or fewer seeds per plant is adequate, 10 plants/mcan be used to obtain about 100 seeds/plant, and 3 plants/m usuallyresults in maximum seed production per plant. Densities of 12 plants/mor less commonly are used for artificial hybridization.

Multiple planting dates about 7 to 14 days apart usually are used tomatch parents of different flowering dates. When differences inflowering dates are extreme between parents, flowering of the laterparent can be hastened by creating an artificially short day orflowering of the earlier parent can be delayed by use of artificiallylong days or delayed planting. For example, crosses with genotypesadapted to the southern U.S. are made in northern U.S. locations bycovering the late genotype with a box, large can, or similar containerto create an artificially short photoperiod of about 12 h for about 15days beginning when there are three nodes with trifoliate leaves on themain stem. Plants induced to flower early tend to have flowers thatself-pollinate when they are small and can be difficult to prepare forhybridization.

Grafting can be used to hasten the flowering of late floweringgenotypes. A scion from a late genotype grafted on a stock that hasbegun to flower will begin to bloom up to 42 days earlier than normal(Kiihl et al., Crop Sci., 17:181-182, 1977). First flowers on the scionappear from 21 to 50 days after the graft.

Observing pod development 7 days after pollination generally is adequateto identify a successful cross. Abortion of pods and seeds can occurseveral weeks after pollination, but the percentage of abortion usuallyis low if plant stress is minimized (Shibles et al., “Soybean,” In: CropPhysiology, Some Case Histories, Evans (ed), Cambridge Univ. Press,Cambridge, England, 51-189, 1975). Pods that develop from artificialhybridization can be distinguished from self-pollinated pods by thepresence of the calyx scar, caused by removal of the sepals. The sepalsbegin to fall off as the pods mature; therefore, harvest should becompleted at or immediately before the time the pods reach their maturecolor. Harvesting pods early also avoids any loss by shattering.

Once harvested, pods are typically air-dried at not more than 38° C.until the seeds contain 13% moisture or less, then the seeds are removedby hand. Seed can be stored satisfactorily at about 25° C. for up to ayear if relative humidity is 50% or less. In humid climates, germinationpercentage declines rapidly unless the seed is dried to 7% moisture andstored in an air-tight container at room temperature. Long-term storagein any climate is best accomplished by drying seed to 7% moisture andstoring it at 10° C. or less in a room maintained at 50% relativehumidity or in an air-tight container.

II. Further Embodiments of the Invention

In certain aspects of the invention, plants of soybean variety D403301are modified to include at least a first desired heritable trait. Suchplants may, in one embodiment, be developed by a plant breedingtechnique called backcrossing, wherein essentially all of themorphological and physiological characteristics of a variety arerecovered in addition to a genetic locus transferred into the plant viathe backcrossing technique. By essentially all of the morphological andphysiological characteristics, it is meant that the characteristics of aplant are recovered that are otherwise present when compared in the sameenvironment, other than occasional variant traits that might ariseduring backcrossing or direct introduction of a transgene. It isunderstood that a locus introduced by backcrossing may or may not betransgenic in origin, and thus the term backcrossing specificallyincludes backcrossing to introduce loci that were created by genetictransformation.

In a typical backcross protocol, the original variety of interest(recurrent parent) is crossed to a second variety (nonrecurrent parent)that carries the single locus of interest to be transferred. Theresulting progeny from this cross are then crossed again to therecurrent parent and the process is repeated until a soybean plant isobtained wherein essentially all of the desired morphological andphysiological characteristics of the recurrent parent are recovered inthe converted plant, in addition to the transferred locus from thenonrecurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a trait or characteristic in the originalvariety. To accomplish this, a locus of the recurrent variety ismodified or substituted with the desired locus from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired morphological and physiologicalconstitution of the original variety. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross; one ofthe major purposes is to add some commercially desirable, agronomicallyimportant trait to the plant. The exact backcrossing protocol willdepend on the characteristic or trait being altered to determine anappropriate testing protocol. Although backcrossing methods aresimplified when the characteristic being transferred is a dominantallele, a recessive allele may also be transferred. In this instance itmay be necessary to introduce a test of the progeny to determine if thedesired characteristic has been successfully transferred.

Soybean varieties can also be developed from more than two parents(Fehr, In: “Soybeans: Improvement, Production and Uses,” 2nd Ed.,Manograph 16:249, 1987). The technique, known as modified backcrossing,uses different recurrent parents during the backcrossing. Modifiedbackcrossing may be used to replace the original recurrent parent with avariety having certain more desirable characteristics or multipleparents may be used to obtain different desirable characteristics fromeach.

Many traits have been identified that are not regularly selected for inthe development of a new inbred but that can be improved by backcrossingtechniques. Traits may or may not be transgenic; examples of thesetraits include, but are not limited to, male sterility, herbicideresistance, resistance to bacterial, fungal, or viral disease, insectand pest resistance, restoration of male fertility, enhanced nutritionalquality, yield stability, and yield enhancement. These comprise genesgenerally inherited through the nucleus.

Direct selection may be applied where the locus acts as a dominanttrait. An example of a dominant trait is the herbicide resistance trait.For this selection process, the progeny of the initial cross are sprayedwith the herbicide prior to the backcrossing. The spraying eliminatesany plants which do not have the desired herbicide resistancecharacteristic, and only those plants which have the herbicideresistance gene are used in the subsequent backcross. This process isthen repeated for all additional backcross generations.

Selection of soybean plants for breeding is not necessarily dependent onthe phenotype of a plant and instead can be based on geneticinvestigations. For example, one may utilize a suitable genetic markerwhich is closely associated with a trait of interest. One of thesemarkers may therefore be used to identify the presence or absence of atrait in the offspring of a particular cross, and hence may be used inselection of progeny for continued breeding. This technique may commonlybe referred to as marker assisted selection. Any other type of geneticmarker or other assay which is able to identify the relative presence orabsence of a trait of interest in a plant may also be useful forbreeding purposes. Procedures for marker assisted selection applicableto the breeding of soybeans are well known in the art. Such methods willbe of particular utility in the case of recessive traits and variablephenotypes, or where conventional assays may be more expensive, timeconsuming or otherwise disadvantageous. Types of genetic markers whichcould be used in accordance with the invention include, but are notnecessarily limited to, Simple Sequence Length Polymorphisms (SSLPs)(Williams et al., Nucleic Acids Res., 18:6531-6535, 1990), RandomlyAmplified Polymorphic DNAs (RAPDs), DNA Amplification Fingerprinting(DAF), Sequence Characterized Amplified Regions (SCARs), ArbitraryPrimed Polymerase Chain Reaction (AP-PCR), Amplified Fragment LengthPolymorphisms (AFLPs) (EP 534 858, specifically incorporated herein byreference in its entirety), and Single Nucleotide Polymorphisms (SNPs)(Wang et al., Science, 280:1077-1082, 1998).

Many qualitative characters also have potential use as phenotype-basedgenetic markers in soybeans; however, some or many may not differ amongvarieties commonly used as parents (Bernard and Weiss, “Qualitativegenetics,” In: Soybeans: Improvement, Production, and Uses, Caldwell(ed), Am. Soc. of Agron., Madison, Wis., 117-154, 1973). The most widelyused genetic markers are flower color (purple dominant to white),pubescence color (brown dominant to gray), and pod color (brown dominantto tan). The association of purple hypocotyl color with purple flowersand green hypocotyl color with white flowers is commonly used toidentify hybrids in the seedling stage. Differences in maturity, height,hilum color, and pest resistance between parents can also be used toverify hybrid plants.

Many useful traits that can be introduced by backcrossing, as well asdirectly into a plant, are those which are introduced by genetictransformation techniques. Genetic transformation may therefore be usedto insert a selected transgene into the soybean variety of the inventionor may, alternatively, be used for the preparation of transgenes whichcan be introduced by backcrossing. Methods for the transformation ofmany economically important plants, including soybeans, are well knownto those of skill in the art. Techniques which may be employed for thegenetic transformation of soybeans include, but are not limited to,electroporation, microprojectile bombardment, Agrobacterium-mediatedtransformation and direct DNA uptake by protoplasts.

To effect transformation by electroporation, one may employ eitherfriable tissues, such as a suspension culture of cells or embryogeniccallus or alternatively one may transform immature embryos or otherorganized tissue directly. In this technique, one would partiallydegrade the cell walls of the chosen cells by exposing them topectin-degrading enzymes (pectolyases) or mechanically wound tissues ina controlled manner.

Protoplasts may also be employed for electroporation transformation ofplants (Bates, Mol. Biotechnol., 2(2):135-145, 1994; Lazzeri, MethodsMol. Biol., 49:95-106, 1995). For example, the generation of transgenicsoybean plants by electroporation of cotyledon-derived protoplasts wasdescribed by Dhir and Widholm in Intl. Pat. App. Publ. No. WO 92/17598,the disclosure of which is specifically incorporated herein byreference.

A particularly efficient method for delivering transforming DNA segmentsto plant cells is microprojectile bombardment. In this method, particlesare coated with nucleic acids and delivered into cells by a propellingforce. Exemplary particles include those comprised of tungsten,platinum, and often, gold. For the bombardment, cells in suspension areconcentrated on filters or solid culture medium. Alternatively, immatureembryos or other target cells may be arranged on solid culture medium.The cells to be bombarded are positioned at an appropriate distancebelow the macroprojectile stopping plate.

An illustrative embodiment of a method for delivering DNA into plantcells by acceleration is the Biolistics Particle Delivery System, whichcan be used to propel particles coated with DNA or cells through ascreen, such as a stainless steel or Nytex screen, onto a surfacecovered with target soybean cells. The screen disperses the particles sothat they are not delivered to the recipient cells in large aggregates.It is believed that a screen intervening between the projectileapparatus and the cells to be bombarded reduces the size of theprojectile aggregate and may contribute to a higher frequency oftransformation by reducing the damage inflicted on the recipient cellsby projectiles that are too large.

Microprojectile bombardment techniques are widely applicable, and may beused to transform virtually any plant species. The application ofmicroprojectile bombardment for the transformation of soybeans isdescribed, for example, in U.S. Pat. No. 5,322,783, the disclosure ofwhich is specifically incorporated herein by reference in its entirety.

Agrobacterium-mediated transfer is another widely applicable system forintroducing gene loci into plant cells. An advantage of the technique isthat DNA can be introduced into whole plant tissues, thereby bypassingthe need for regeneration of an intact plant from a protoplast. ModernAgrobacterium transformation vectors are capable of replication in E.coli as well as Agrobacterium, allowing for convenient manipulations(Klee et al., Bio. Tech., 3(7):637-642, 1985). Moreover, recenttechnological advances in vectors for Agrobacterium-mediated genetransfer have improved the arrangement of genes and cloning sites in thevectors to facilitate the construction of vectors capable of expressingvarious polypeptide coding genes. Vectors can have convenientmultiple-cloning sites (MCS) flanked by a promoter and a polyadenylationsite for direct expression of inserted polypeptide coding genes. Othervectors can comprise site-specific recombination sequences, enablinginsertion of a desired DNA sequence without the use of restrictionenzymes (Curtis et al., Plant Physiology 133:462-469, 2003).Additionally, Agrobacterium containing both armed and disarmed Ti genescan be used for transformation.

In those plant strains where Agrobacterium-mediated transformation isefficient, it is the method of choice because of the facile and definednature of the gene locus transfer. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art (Fraley et al., Bio. Tech., 3(7):629-635, 1985; U.S.Pat. No. 5,563,055). Use of Agrobacterium in the context of soybeantransformation has been described, for example, by Chee and Slightom(Methods Mol. Biol., 44:101-119, 1995) and in U.S. Pat. No. 5,569,834,the disclosures of which are specifically incorporated herein byreference in their entirety.

Transformation of plant protoplasts also can be achieved using methodsbased on calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments (see, e.g.,Potrykus et al., Mol. Gen. Genet., 199(2):169-177, 1985; Omirulleh etal., Plant Mol. Biol., 21(3):415-428, 1993; Fromm et al., Nature,319(6056):791-793, 1986; Uchimiya et al., Mol. Gen. Genet.,204(2):204-207, 1986; Marcotte et al., Nature, 335(6189):454-457, 1988).The demonstrated ability to regenerate soybean plants from protoplastsmakes each of these techniques applicable to soybean (Dhir et al., PlantCell Rep., 10(2):97-101, 1991).

Many hundreds if not thousands of different genes are known and couldpotentially be introduced into a soybean plant according to theinvention. Non-limiting examples of particular genes and correspondingphenotypes one may choose to introduce into a soybean plant arepresented below.

A. Herbicide Resistance

Numerous herbicide resistance genes are known and may be employed withthe invention. An example is a gene conferring resistance to a herbicidethat inhibits the growing point or meristem, such as an imidazalinone ora sulfonylurea. Exemplary genes in this category code for mutant ALS andAHAS enzyme as described, for example, by Lee et al., EMBO J., 7:1241,1988; Gleen et al., Plant Molec. Biology, 18:1185-1187, 1992; and Mikiet al., Theor. Appl. Genet., 80:449, 1990.

Resistance genes for glyphosate (resistance conferred by mutant5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyltransferase (PAT) and Streptomyces hygroscopicusphosphinothricin-acetyl transferase (bar) genes) may also be used. See,for example, U.S. Pat. No. 4,940,835 to Shah et al., which discloses thenucleotide sequence of a form of EPSPS which can confer glyphosateresistance. Examples of specific EPSPS transformation events conferringglyphosate resistance are provided by U.S. Pat. Nos. 6,040,497 and7,632,985. The MON89788 event disclosed in U.S. Pat. No. 7,632,985 inparticular is beneficial in conferring glyphosate tolerance incombination with an increase in average yield relative to prior events.

A DNA molecule encoding a mutant aroA gene can be obtained under ATCCAccession Number 39256, and the nucleotide sequence of the mutant geneis disclosed in U.S. Pat. No. 4,769,061 to Comai. A hygromycin Bphosphotransferase gene from E. coli which confers resistance toglyphosate in tobacco callus and plants is described in Penaloza-Vazquezet al., Plant Cell Reports, 14:482-487, 1995. European PatentApplication No. 0 333 033 to Kumada et al., and U.S. Pat. No. 4,975,374to Goodman et al., disclose nucleotide sequences of glutamine synthetasegenes which confer resistance to herbicides such as L-phosphinothricin.The nucleotide sequence of a phosphinothricin acetyltransferase gene isprovided in European Patent Application No. 0 242 246 to Leemans et al.DeGreef et al. (Biotechnology, 7:61, 1989), describe the production oftransgenic plants that express chimeric bar genes coding forphosphinothricin acetyl transferase activity. Exemplary genes conferringresistance to phenoxy propionic acids and cyclohexanediones, such assethoxydim and haloxyfop are the Acct-S1, Acct-S2 and Acct-S3 genesdescribed by Marshall et al., (Theor. Appl. Genet., 83:4:35, 1992).

Genes are also known conferring resistance to a herbicide that inhibitsphotosynthesis, such as a triazine (psbA and gs+genes) and abenzonitrile (nitrilase gene). Przibila et al. (Plant Cell, 3:169, 1991)describe the transformation of Chlamydomonas with plasmids encodingmutant psbA genes. Nucleotide sequences for nitrilase genes aredisclosed in U.S. Pat. No. 4,810,648 to Stalker, and DNA moleculescontaining these genes are available under ATCC Accession Nos. 53435,67441, and 67442. Cloning and expression of DNA coding for a glutathioneS-transferase is described by Hayes et al. (Biochem. J., 285(Pt1):173-180, 1992). Protoporphyrinogen oxidase (PPO) is the target of thePPO-inhibitor class of herbicides; a PPO-inhibitor resistant PPO genewas recently identified in Amaranthus tuberculatus (Patzoldt et al.,PNAS, 103(33):12329-2334, 2006). The herbicide methyl viologen inhibitsCO₂ assimilation. Foyer et al. (Plant Physiol., 109:1047-1057, 1995)describe a plant overexpressing glutathione reductase (GR) which isresistant to methyl viologen treatment.

Siminszky (Phytochemistry Reviews, 5:445-458, 2006) describes plantcytochrome P450-mediated detoxification of multiple, chemicallyunrelated classes of herbicides. Modified bacterial genes have beensuccessfully demonstrated to confer resistance to atrazine, a herbicidethat binds to the plastoquinone-binding membrane protein Q_(B) inphotosystem II to inhibit electron transport. See, for example, studiesby Cheung et al. (PNAS, 85(2):391-395, 1988), describing tobacco plantsexpressing the chloroplast psbA gene from an atrazine-resistant biotypeof Amaranthus hybridus fused to the regulatory sequences of a nucleargene, and Wang et al. (Plant Biotech. J., 3:475-486, 2005), describingtransgenic alfalfa, Arabidopsis, and tobacco plants expressing the atzAgene from Pseudomonas sp. that were able to detoxify atrazine.

Bayley et al. (Theor. Appl. Genet., 83:645-649, 1992) describe thecreation of 2,4-D-resistant transgenic tobacco and cotton plants usingthe 2,4-D monooxygenase gene tfdA from Alcaligenes eutrophus plasmidpJP5. U.S. Pat. App. Pub. No. 20030135879 describes the isolation of agene for dicamba monooxygenase (DMO) from Psueodmonas maltophilia thatis involved in the conversion of dicamba to a non-toxic3,6-dichlorosalicylic acid and thus may be used for producing plantstolerant to this herbicide.

Other examples of herbicide resistance have been described, forinstance, in U.S. Pat. Nos. 6,803,501; 6,448,476; 6,248,876; 6,225,114;6,107,549; 5,866,775; 5,804,425; 5,633,435; 5,463,175.

B. Disease and Pest Resistance

Plant defenses are often activated by specific interaction between theproduct of a disease resistance gene (R) in the plant and the product ofa corresponding avirulence (Avr) gene in the pathogen. A plant line canbe transformed with cloned resistance gene to engineer plants that areresistant to specific pathogen strains. See, for example Jones et al.(Science, 266:7891, 1994) (cloning of the tomato Cf-9 gene forresistance to Cladosporium fulvum); Martin et al. (Science, 262: 1432,1993) (tomato Pto gene for resistance to Pseudomonas syringae pv.tomato); and Mindrinos et al. (Cell, 78(6):1089-1099, 1994) (ArabidopsisRPS2 gene for resistance to Pseudomonas syringae).

A viral-invasive protein or a complex toxin derived therefrom may alsobe used for viral disease resistance. For example, the accumulation ofviral coat proteins in transformed plant cells imparts resistance toviral infection and/or disease development effected by the virus fromwhich the coat protein gene is derived, as well as by related viruses.See Beachy et al. (Ann. Rev. Phytopathol., 28:451, 1990). Coatprotein-mediated resistance has been conferred upon transformed plantsagainst alfalfa mosaic virus, cucumber mosaic virus, tobacco streakvirus, potato virus X, potato virus Y, tobacco etch virus, tobaccorattle virus and tobacco mosaic virus. Id.

A virus-specific antibody may also be used. See, for example,Tavladoraki et al. (Nature, 366:469, 1993), who show that transgenicplants expressing recombinant antibody genes are protected from virusattack. Virus resistance has also been described in, for example, U.S.Pat. Nos. 6,617,496; 6,608,241; 6,015,940; 6,013,864; 5,850,023 and5,304,730. Additional means of inducing whole-plant resistance to apathogen include modulation of the systemic acquired resistance (SAR) orpathogenesis related (PR) genes, for example genes homologous to theArabidopsis thaliana NIM1/NPR1/SAI1, and/or by increasing salicylic acidproduction (Ryals et al., Plant Cell, 8:1809-1819, 1996).

Logemann et al. (Biotechnology, 10:305, 1992), for example, disclosetransgenic plants expressing a barley ribosome-inactivating gene thathave an increased resistance to fungal disease. Plant defensins may beused to provide resistance to fungal pathogens (Thomma et al., Planta,216:193-202, 2002). Other examples of fungal disease resistance areprovided in U.S. Pat. Nos. 6,653,280; 6,573,361; 6,506,962; 6,316,407;6,215,048; 5,516,671; 5,773,696; 6,121,436; and 6,316,407.

Nematode resistance has been described, for example, in U.S. Pat. No.6,228,992, and bacterial disease resistance has been described in U.S.Pat. No. 5,516,671.

The use of the herbicide glyphosate for disease control in soybeanplants containing event MON89788, which confers glyphosate tolerance,has also been described in U.S. Pat. No. 7,608,761.

C. Insect Resistance

One example of an insect resistance gene includes a Bacillusthuringiensis protein, a derivative thereof or a synthetic polypeptidemodeled thereon. See, for example, Geiser et al. (Gene, 48(1):109-118,1986), who disclose the cloning and nucleotide sequence of a Bacillusthuringiensis δ-endotoxin gene. Moreover, DNA molecules encodingδ-endotoxin genes can be purchased from the American Type CultureCollection, Manassas, Va., for example, under ATCC Accession Nos. 40098,67136, 31995 and 31998. Another example is a lectin. See, for example,Van Damme et al., (Plant Molec. Biol., 24:25, 1994), who disclose thenucleotide sequences of several Clivia miniata mannose-binding lectingenes. A vitamin-binding protein may also be used, such as avidin. SeePCT Application No. US93/06487, the contents of which are herebyincorporated by reference. This application teaches the use of avidinand avidin homologues as larvicides against insect pests.

Yet another insect resistance gene is an enzyme inhibitor, for example,a protease or proteinase inhibitor or an amylase inhibitor. See, forexample, Abe et al. (J. Biol. Chem., 262:16793, 1987) (nucleotidesequence of rice cysteine proteinase inhibitor), Huub et al. (PlantMolec. Biol., 21:985, 1993) (nucleotide sequence of cDNA encodingtobacco proteinase inhibitor I), and Sumitani et al. (Biosci. Biotech.Biochem., 57:1243, 1993) (nucleotide sequence of Streptomycesnitrosporeus α-amylase inhibitor).

An insect-specific hormone or pheromone may also be used. See, forexample, the disclosure by Hammock et al. (Nature, 344:458, 1990), ofbaculovirus expression of cloned juvenile hormone esterase, aninactivator of juvenile hormone; Gade and Goldsworthy (Eds.Physiological System in Insects, Elsevier Academic Press, Burlington,Mass., 2007), describing allostatins and their potential use in pestcontrol; and Palli et al. (Vitam. Horm., 73:59-100, 2005), disclosinguse of ecdysteroid and ecdysteroid receptor in agriculture. The diuretichormone receptor (DHR) was identified in Price et al. (Insect Mol.Biol., 13:469-480, 2004) as a candidate target of insecticides.

Still other examples include an insect-specific antibody or animmunotoxin derived therefrom and a developmental-arrestive protein. SeeTaylor et al. (Seventh Inn Symposium on Molecular Plant-MicrobeInteractions, Edinburgh, Scotland, Abstract W97, 1994), who describedenzymatic inactivation in transgenic tobacco via production ofsingle-chain antibody fragments. Numerous other examples of insectresistance have been described. See, for example, U.S. Pat. Nos.6,809,078; 6,713,063; 6,686,452; 6,657,046; 6,645,497; 6,642,030;6,639,054; 6,620,988; 6,593,293; 6,555,655; 6,538,109; 6,537,756;6,521,442; 6,501,009; 6,468,523; 6,326,351; 6,313,378; 6,284,949;6,281,016; 6,248,536; 6,242,241; 6,221,649; 6,177,615; 6,156,573;6,153,814; 6,110,464; 6,093,695; 6,063,756; 6,063,597; 6,023,013;5,959,091; 5,942,664; 5,942,658, 5,880,275; 5,763,245 and 5,763,241.

D. Male Sterility

Genetic male sterility is available in soybeans and, although notrequired for crossing soybean plants, can increase the efficiency withwhich hybrids are made, in that it can eliminate the need to physicallyemasculate the soybean plant used as a female in a given cross. (Brimand Stuber, Crop Sci., 13:528-530, 1973). Herbicide-inducible malesterility systems have also been described. (U.S. Pat. No. 6,762,344).

Where one desires to employ male-sterility systems, it may be beneficialto also utilize one or more male-fertility restorer genes. For example,where cytoplasmic male sterility (CMS) is used, hybrid seed productionrequires three inbred lines: (1) a cytoplasmically male-sterile linehaving a CMS cytoplasm; (2) a fertile inbred with normal cytoplasm,which is isogenic with the CMS line for nuclear genes (“maintainerline”); and (3) a distinct, fertile inbred with normal cytoplasm,carrying a fertility restoring gene (“restorer” line). The CMS line ispropagated by pollination with the maintainer line, with all of theprogeny being male sterile, as the CMS cytoplasm is derived from thefemale parent. These male sterile plants can then be efficientlyemployed as the female parent in hybrid crosses with the restorer line,without the need for physical emasculation of the male reproductiveparts of the female parent.

The presence of a male-fertility restorer gene results in the productionof fully fertile F₁ hybrid progeny. If no restorer gene is present inthe male parent, male-sterile hybrids are obtained. Such hybrids areuseful where the vegetative tissue of the soybean plant is utilized, butin many cases the seeds will be deemed the most valuable portion of thecrop, so fertility of the hybrids in these crops must be restored.Therefore, one aspect of the current invention concerns plants of thesoybean variety D403301 comprising a genetic locus capable of restoringmale fertility in an otherwise male-sterile plant. Examples ofmale-sterility genes and corresponding restorers which could be employedwith the plants of the invention are well known to those of skill in theart of plant breeding (see, e.g., U.S. Pat. Nos. 5,530,191 and5,684,242, the disclosures of which are each specifically incorporatedherein by reference in their entirety).

E. Modified Fatty Acid, Phytate and Carbohydrate Metabolism

Genes may be used conferring modified fatty acid metabolism. Forexample, stearyl-ACP desaturase genes may be used. See Knutzon et al.(Proc. Natl. Acad. Sci. USA, 89:2624, 1992). Various fatty aciddesaturases have also been described. McDonough et al. describe aSaccharomyces cerevisiae OLE1 gene encoding Δ9-fatty acid desaturase, anenzyme which forms the monounsaturated palmitoleic (16:1) and oleic(18:1) fatty acids from palmitoyl (16:0) or stearoyl (18:0) CoA (J.Biol. Chem., 267(9):5931-5936, 1992). Fox et al. describe a geneencoding a stearoyl-acyl carrier protein delta-9 desaturase from castor(Proc. Natl. Acad. Sci. USA, 90(6):2486-2490, 1993). Reddy et al.describe Δ6- and Δ2-desaturases from the cyanobacteria Synechocystisresponsible for the conversion of linoleic acid (18:2) togamma-linolenic acid (18:3 gamma) (Plant Mol. Biol., 22(2):293-300,1993). A gene from Arabidopsis thaliana that encodes an omega-3desaturase has been identified (Arondel et al. Science,258(5086):1353-1355, 1992). Plant Δ9-desaturases (PCT Application Publ.No. WO 91/13972) and soybean and Brassica Δ15-desaturases (EuropeanPatent Application Publ. No. EP 0616644) have also been described. U.S.Pat. No. 7,622,632 describes fungal Δ15-desaturases and their use inplants. EP Patent No. 1656449 describes Δ6-desaturases from Primula aswell as soybean plants having an increased stearidonic acid (SDA, 18:4)content. U.S. Pat. App. Pub. No. 2008-0260929 describes expression oftransgenic desaturase enzymes in corn plants, and improved fatty acidprofiles resulting therefrom.

Modified oil production is disclosed, for example, in U.S. Pat. Nos.6,444,876; 6,426,447 and 6,380,462. High oil production is disclosed,for example, in U.S. Pat. Nos. 6,495,739; 5,608,149; 6,483,008 and6,476,295. Modified fatty acid content is disclosed, for example, inU.S. Pat. Nos. 6,828,475; 6,822,141; 6,770,465; 6,706,950; 6,660,849;6,596,538; 6,589,767; 6,537,750; 6,489,461 and 6,459,018.

Phytate metabolism may also be modified by introduction of aphytase-encoding gene to enhance breakdown of phytate, adding more freephosphate to the transformed plant. For example, see Van Hartingsveldtet al. (Gene, 127:87, 1993), for a disclosure of the nucleotide sequenceof an Aspergillus niger phytase gene. In soybean, this, for example,could be accomplished by cloning and then reintroducing DNA associatedwith the single allele which is responsible for soybean mutantscharacterized by low levels of phytic acid. See Raboy et al. (PlantPhysiol., 124(1):355-368, 2000).

A number of genes are known that may be used to alter carbohydratemetabolism. For example, plants may be transformed with a gene codingfor an enzyme that alters the branching pattern of starch. See Shirozaet al. (J. Bacteriol., 170:810, 1988) (nucleotide sequence ofStreptococcus mutans fructosyltransferase gene), Steinmetz et al. (Mol.Gen. Genet., 20:220, 1985) (nucleotide sequence of Bacillus subtilislevansucrase gene), Pen et al. (Biotechnology, 10:292, 1992) (productionof transgenic plants that express Bacillus licheniformis α-amylase),Elliot et al. (Plant Molec. Biol., 21:515, 1993) (nucleotide sequencesof tomato invertase genes), Sergaard et al. (J. Biol. Chem., 268:22480,1993) (site-directed mutagenesis of barley α-amylase gene), and Fisheret al. (Plant Physiol., 102:1045, 1993) (maize endosperm starchbranching enzyme II). The Z10 gene encoding a 10 kD zein storage proteinfrom maize may also be used to alter the quantities of 10 kD zein in thecells relative to other components (Kirihara et al., Gene,71(2):359-370, 1988).

F. Resistance to Abiotic Stress

Abiotic stress includes dehydration or other osmotic stress, salinity,high or low light intensity, high or low temperatures, submergence,exposure to heavy metals, and oxidative stress.Delta-pyrroline-5-carboxylate synthetase (P5CS) from mothbean has beenused to provide protection against general osmotic stress.Mannitol-1-phosphate dehydrogenase (mt1D) from E. coli has been used toprovide protection against drought and salinity. Choline oxidase (codAfrom Arthrobactor globiformis) can protect against cold and salt. E.coli choline dehydrogenase (betA) provides protection against salt.Additional protection from cold can be provided by omega-3-fatty aciddesaturase (fad7) from Arabidopsis thaliana. Trehalose-6-phosphatesynthase and levan sucrase (SacB) from yeast and Bacillus subtilis,respectively, can provide protection against drought (summarized fromAnnex II Genetic Engineering for Abiotic Stress Tolerance in Plants,Consultative Group On International Agricultural Research TechnicalAdvisory Committee). Overexpression of superoxide dismutase can be usedto protect against superoxides, as described in U.S. Pat. No. 5,538,878to Thomas et al.

G. Additional Traits

Additional traits can be introduced into the soybean variety of thepresent invention. A non-limiting example of such a trait is a codingsequence which decreases RNA and/or protein levels. The decreased RNAand/or protein levels may be achieved through RNAi methods, such asthose described in U.S. Pat. No. 6,506,559 to Fire and Mellow.

Another trait that may find use with the soybean variety of theinvention is a sequence which allows for site-specific recombination.Examples of such sequences include the FRT sequence, used with the FLPrecombinase (Zhu and Sadowski, J. Biol. Chem., 270:23044-23054, 1995);and the LOX sequence, used with CRE recombinase (Sauer, Mol. Cell.Biol., 7:2087-2096, 1987). The recombinase genes can be encoded at anylocation within the genome of the soybean plant, and are active in thehemizygous state.

It may also be desirable to make soybean plants more tolerant to or moreeasily transformed with Agrobacterium tumefaciens. Expression of p53 andiap, two baculovirus cell-death suppressor genes, inhibited tissuenecrosis and DNA cleavage. Additional targets can include plant-encodedproteins that interact with the Agrobacterium Vir genes; enzymesinvolved in plant cell wall formation; and histones, histoneacetyltransferases and histone deacetylases (reviewed in Gelvin,Microbiology & Mol. Biol. Reviews, 67:16-37, 2003).

In addition to the modification of oil, fatty acid or phytate contentdescribed above, it may additionally be beneficial to modify the amountsor levels of other compounds. For example, the amount or composition ofantioxidants can be altered. See, for example, U.S. Pat. No. 6,787,618;U.S. Pat. App. Pub. No. 20040034886 and International Patent App. Pub.No. WO 00/68393, which disclose the manipulation of antioxidant levels,and International Patent App. Pub. No. WO 03/082899, which discloses themanipulation of a antioxidant biosynthetic pathway.

Additionally, seed amino acid content may be manipulated. U.S. Pat. No.5,850,016 and International Patent App. Pub. No. WO 99/40209 disclosethe alteration of the amino acid compositions of seeds. U.S. Pat. Nos.6,080,913 and 6,127,600 disclose methods of increasing accumulation ofessential amino acids in seeds.

U.S. Pat. No. 5,559,223 describes synthetic storage proteins in whichthe levels of essential amino acids can be manipulated. InternationalPatent App. Pub. No. WO 99/29882 discloses methods for altering aminoacid content of proteins. International Patent App. Pub. No. WO 98/20133describes proteins with enhanced levels of essential amino acids.International Patent App. Pub. No. WO 98/56935 and U.S. Pat. Nos.6,346,403, 6,441,274 and 6,664,445 disclose plant amino acidbiosynthetic enzymes. International Patent App. Pub. No. WO 98/45458describes synthetic seed proteins having a higher percentage ofessential amino acids than wild-type.

U.S. Pat. No. 5,633,436 discloses plants comprising a higher content ofsulfur-containing amino acids; U.S. Pat. No. 5,885,801 discloses plantscomprising a high threonine content; U.S. Pat. Nos. 5,885,802 and5,912,414 disclose plants comprising a high methionine content; U.S.Pat. No. 5,990,389 discloses plants comprising a high lysine content;U.S. Pat. No. 6,459,019 discloses plants comprising an increased lysineand threonine content; International Patent App. Pub. No. WO 98/42831discloses plants comprising a high lysine content; International PatentApp. Pub. No. WO 96/01905 discloses plants comprising a high threoninecontent and International Patent App. Pub. No. WO 95/15392 disclosesplants comprising a high lysine content.

III. Origin and Breeding History of an Exemplary Single Locus ConvertedPlant

It is known to those of skill in the art that, by way of the techniqueof backcrossing, one or more traits may be introduced into a givenvariety while otherwise retaining essentially all of the traits of thatvariety. An example of such backcrossing to introduce a trait into astarting variety is described in U.S. Pat. No. 6,140,556, the entiredisclosure of which is specifically incorporated herein by reference.The procedure described in U.S. Pat. No. 6,140,556 can be summarized asfollows: The soybean variety known as Williams '82 [Glycine max L.Merr.] (Reg. No. 222, PI 518671) was developed using backcrossingtechniques to transfer a locus comprising the Rps₁ gene to the varietyWilliams (Bernard and Cremeens, Crop Sci., 28:1027-1028, 1988). Williams'82 is a composite of four resistant lines from the BC₆F₃ generation,which were selected from 12 field-tested resistant lines fromWilliams×Kingwa. The variety Williams was used as the recurrent parentin the backcross and the variety Kingwa was used as the source of theRps₁, locus. This gene locus confers resistance to 19 of the 24 races ofthe fungal agent phytopthora root rot.

The F₁ or F₂ seedlings from each backcross round were tested forresistance to the fungus by hypocotyl inoculation using the inoculum ofrace 5. The final generation was tested using inoculum of races 1 to 9.In a backcross such as this, where the desired characteristic beingtransferred to the recurrent parent is controlled by a major gene whichcan be readily evaluated during the backcrossing, it is common toconduct enough backcrosses to avoid testing individual progeny forspecific traits such as yield in extensive replicated tests. In general,four or more backcrosses are used when there is no evaluation of theprogeny for specific traits, such as yield. As in this example, lineswith the phenotype of the recurrent parent may be composited without theusual replicated tests for traits such as yield, protein or oilpercentage in the individual lines.

The variety Williams '82 is comparable to the recurrent parent varietyWilliams in its traits except resistance to phytopthora rot. Forexample, both varieties have a relative maturity of 38, indeterminatestems, white flowers, brown pubescence, tan pods at maturity and shinyyellow seeds with black to light black hila.

IV. Tissue Cultures and in vitro Regeneration of Soybean Plants

A further aspect of the invention relates to tissue cultures of thesoybean variety designated D403301. As used herein, the term “tissueculture” indicates a composition comprising isolated cells of the sameor a different type or a collection of such cells organized into partsof a plant. Exemplary types of tissue cultures are protoplasts, calliand plant cells that are intact in plants or parts of plants, such asembryos, pollen, flowers, leaves, roots, root tips, anthers, and thelike. In one embodiment, the tissue culture comprises embryos,protoplasts, meristematic cells, pollen, leaves or anthers.

Exemplary procedures for preparing tissue cultures of regenerablesoybean cells and regenerating soybean plants therefrom, are disclosedin U.S. Pat. Nos. 4,992,375; 5,015,580; 5,024,944 and 5,416,011, each ofthe disclosures of which is specifically incorporated herein byreference in its entirety.

An important ability of a tissue culture is the capability to regeneratefertile plants. This allows, for example, transformation of the tissueculture cells followed by regeneration of transgenic plants. Fortransformation to be efficient and successful, DNA must be introducedinto cells that give rise to plants or germ-line tissue.

Soybeans typically are regenerated via two distinct processes: shootmorphogenesis and somatic embryogenesis (Finer, Cheng, Verma, “Soybeantransformation: Technologies and progress,” In: Soybean: Genetics,Molecular Biology and Biotechnology, CAB Intl, Verma and Shoemaker (ed),Wallingford, Oxon, UK, 250-251, 1996). Shoot morphogenesis is theprocess of shoot meristem organization and development. Shoots grow outfrom a source tissue and are excised and rooted to obtain an intactplant. During somatic embryogenesis, an embryo (similar to the zygoticembryo), containing both shoot and root axes, is formed from somaticplant tissue. An intact plant rather than a rooted shoot results fromthe germination of the somatic embryo.

Shoot morphogenesis and somatic embryogenesis are different processesand the specific route of regeneration is primarily dependent on theexplant source and media used for tissue culture manipulations. Whilethe systems are different, both systems show variety-specific responseswhere some lines are more responsive to tissue culture manipulationsthan others. A line that is highly responsive in shoot morphogenesis maynot generate many somatic embryos. Lines that produce large numbers ofembryos during an ‘induction’ step may not give rise to rapidly-growingproliferative cultures. Therefore, it may be desired to optimize tissueculture conditions for each soybean line. These optimizations mayreadily be carried out by one of skill in the art of tissue culturethrough small-scale culture studies. In addition to line-specificresponses, proliferative cultures can be observed with both shootmorphogenesis and somatic embryogenesis. Proliferation is beneficial forboth systems, as it allows a single, transformed cell to multiply to thepoint that it will contribute to germ-line tissue.

Shoot morphogenesis was first reported by Wright et al. (Plant CellReports, 5:150-154, 1986) as a system whereby shoots were obtained denovo from cotyledonary nodes of soybean seedlings. The shoot meristemswere formed subepidermally and morphogenic tissue could proliferate on amedium containing benzyl adenine (BA). This system can be used fortransformation if the subepidermal, multicellular origin of the shootsis recognized and proliferative cultures are utilized. The idea is totarget tissue that will give rise to new shoots and proliferate thosecells within the meristematic tissue to lessen problems associated withchimerism. Formation of chimeras, resulting from transformation of onlya single cell in a meristem, are problematic if the transformed cell isnot adequately proliferated and does not does not give rise to germ-linetissue. Once the system is well understood and reproducedsatisfactorily, it can be used as one target tissue for soybeantransformation.

Somatic embryogenesis in soybean was first reported by Christianson etal. (Science, 222:632-634, 1983) as a system in which embryogenic tissuewas initially obtained from the zygotic embryo axis. These embryogeniccultures were proliferative but the repeatability of the system was lowand the origin of the embryos was not reported. Later histologicalstudies of a different proliferative embryogenic soybean culture showedthat proliferative embryos were of apical or surface origin with a smallnumber of cells contributing to embryo formation. The origin of primaryembryos (the first embryos derived from the initial explant) isdependent on the explant tissue and the auxin levels in the inductionmedium (Hartweck et al., In Vitro Cell. Develop. Bio., 24:821-828,1988). With proliferative embryonic cultures, single cells or smallgroups of surface cells of the ‘older’ somatic embryos form the ‘newer’embryos.

Embryogenic cultures can also be used successfully for regeneration,including regeneration of transgenic plants, if the origin of theembryos is recognized and the biological limitations of proliferativeembryogenic cultures are understood. Biological limitations include thedifficulty in developing proliferative embryogenic cultures and reducedfertility problems (culture-induced variation) associated with plantsregenerated from long-term proliferative embryogenic cultures. Some ofthese problems are accentuated in prolonged cultures. The use of morerecently cultured cells may decrease or eliminate such problems.

V. Definitions

In the description and tables, a number of terms are used. In order toprovide a clear and consistent understanding of the specification andclaims, the following definitions are provided:

A: When used in conjunction with the word “comprising” or other openlanguage in the claims, the words “a” and “an” denote “one or more.”

About: Refers to embodiments or values that include the standarddeviation of the mean for a given item being measured.

Allele: Any of one or more alternative forms of a gene locus, all ofwhich relate to one trait or characteristic. In a diploid cell ororganism, the two alleles of a given gene occupy corresponding loci on apair of homologous chromosomes.

Aphids: Aphid resistance in greenhouse screening is scored on a scalefrom 1 to 9 based on foliar symptoms and number of aphids; Resistant (R)corresponds to a rating of 1 -3.9, moderately resistant (MR) 4.0 -5.9,moderately susceptible to moderately resistant (MS-MR) 6.0 -6.9, andsusceptible (S) 7.0 -9.0.

Asian Soybean Rust (ASR): ASR may be visually scored from 1 to 5, where1=immune; 2=leaf exhibits red/brown lesions over less than 50% ofsurface; 3=leaf exhibits red/brown lesions over greater than 50% ofsurface; 4=leaf exhibits tan lesions over less than 50% of surface; and5=leaf exhibits tan lesions over greater than 50% of surface. Resistanceto ASR may be characterized phenotypically as well as genetically.Soybean plants phenotypically characterized as resistant to ASRtypically exhibit red brown lesions covering less than 25% of the leaf.Genetic characterization of ASR resistance may be carried out, forexample, by identifying the presence in a soybean plant of one or moregenetic markers linked to the ASR resistance.

Backcrossing: A process in which a breeder repeatedly crosses hybridprogeny, for example a first generation hybrid (F₁), back to one of theparents of the hybrid progeny. Backcrossing can be used to introduce oneor more single locus conversions from one genetic background intoanother.

Brown Stem Rot (BSR): The greenhouse score is based on the incidence andseverity of pith discoloration. Scores are converted to a 1-9 scalewhere Resistant (R) corresponds to a rating <3.1, moderately resistant(MR) 3.1-5.0, moderately susceptible (MS) 5.1-6.9, susceptible (S)7.0-7.9, and highly susceptible (HS) >7.9.

Chloride Sensitivity: Plants may be categorized as “includers” or“excluders” with respect to chloride sensitivity. Excluders tend topartition chloride in the root systems and reduce the amount of chloridetransported to more sensitive, aboveground tissues. Therefore excludersmay display increased tolerance to elevated soil chloride levelscompared to includers. Greenhouse screening Chloride tolerance isreported on a 1-9 scale where a rating less than 3 is considered andexcluder and 4-9 is considered an includer.

Chromatography: A technique wherein a mixture of dissolved substancesare bound to a solid support followed by passing a column of fluidacross the solid support and varying the composition of the fluid. Thecomponents of the mixture are separated by selective elution.

Crossing: The mating of two parent plants.

Cross-pollination: Fertilization by the union of two gametes fromdifferent plants.

Emasculate: The removal of plant male sex organs or the inactivation ofthe organs with a cytoplasmic or nuclear genetic factor or a chemicalagent conferring male sterility.

Emergence (EMR): The emergence score describes the ability of a seed toemerge from the soil after planting. Each genotype is given a 1 to 9score based on its percent of emergence. A score of 1 indicates anexcellent rate and percent of emergence, an intermediate score of 5indicates an average rating and a 9 score indicates a very poor rate andpercent of emergence.

Enzymes: Molecules which can act as catalysts in biological reactions.

F₁ Hybrid: The first generation progeny of the cross of two nonisogenicplants.

Fatty Acids: Are measured and reported as a percent of the total oilcontent. In addition to the typical composition of fatty acids incommodity soybeans, some soybean varieties have modified profiles. Lowlinolenic acid soybean oil as defined herein contains 3% or lesslinolenic acid. Mid oleic acid soybean oil as defined herein containstypically 50-60% oleic acid. High oleic soybean oil as defined hereincontains typically 75% or greater oleic acid. Stearidonic acid levelsare typically 0 percent in commodity soybeans.

Frog Eye Leaf Spot (FELS): Greenhouse assay reaction scores are based onfoliar symptom severity, measured using a 1-9 scale. Resistant (R)corresponds to a rating <3, moderately resistant (MR) 3.0-4.9,moderately susceptible (MS) 5.0-6.9, and susceptible (S) >6.9.

Genotype: The genetic constitution of a cell or organism.

Haploid: A cell or organism having one set of the two sets ofchromosomes in a diploid.

Iron-Deficiency Chlorosis (IDE=early; IDL=late): Iron-deficiencychlorosis is scored in a system ranging from 1 to 9 based on visualobservations. A score of 1 means no stunting of the plants or yellowingof the leaves and a score of 9 indicates the plants are dead or dyingcaused by iron-deficiency chlorosis; a score of 5 means plants haveintermediate health with some leaf yellowing.

Linkage: A phenomenon wherein alleles on the same chromosome tend tosegregate together more often than expected by chance if theirtransmission was independent.

Linolenic Acid Content (LLN): Low-linolenic acid soybean oil containsthree percent or less linolenic acid, compared to eight percentlinolenic acid for traditional soybeans.

Lodging Resistance (LDG): Lodging is rated on a scale of 1 to 9. A scoreof 1 indicates erect plants. A score of 5 indicates plants are leaningat a 45 degree(s) angle in relation to the ground and a score of 9indicates plants are lying on the ground.

Marker: A readily detectable phenotype, preferably inherited incodominant fashion (both alleles at a locus in a diploid heterozygoteare readily detectable), with no environmental variance component, i.e.,heritability of 1.

Maturity Date (MAT): Plants are considered mature when 95% of the podshave reached their mature color. The maturity date is typicallydescribed in measured days after August 31 in the northern hemisphere.

Moisture (MST): The average percentage moisture in the seeds of thevariety.

Oil or Oil Percent: Seed oil content is measured and reported on apercentage basis.

Or: As used herein is meant to mean “and/or” and be interchangeabletherewith unless explicitly indicated to refer to the alternative only.

Phenotype: The detectable characteristics of a cell or organism, whichcharacteristics are the manifestation of gene expression.

Phenotypic Score (PSC): The phenotypic score is a visual rating of thegeneral appearance of the variety. All visual traits are considered inthe score, including healthiness, standability, appearance and freedomfrom disease. Ratings are scored as 1 being poor to 9 being excellent.

Phytophthora Root Rot (PRR): Disorder in which the most recognizablesymptom is stem rot. Brown discoloration ranges below the soil line andup to several inches above the soil line. Leaves often turn yellow, dullgreen and/or gray and may become brown and wilted, but remain attachedto the plant.

Phytophthora Allele: Susceptibility or resistance to Phytophthora rootrot races is affected by alleles such as Rps1a (denotes resistance toRaces 1, 2, 10, 11, 13-18, 24, 26, 27, 31, 32, and 36); Rps1c (denotesresistance to Races 1-3, 6-11, 13, 15, 17, 21, 23, 24, 26, 28-30, 32, 34and 36); Rps1k (denotes resistance to Races 1-11, 13-15, 17, 18, 21-24,26, 36 and 37); Rps2 (denotes resistance to Races 1-5, 9-29, 33, 34 and36-39); Rps3a (denotes resistance to Races 1-5, 8, 9, 11, 13, 14, 16,18, 23, 25, 28, 29, 31-35); Rps6 (denotes resistance to Races 1-4, 10,12, 14-16, 18-21 and 25); and Rps7 (denotes resistance to Races 2, 12,16, 18, 19, 33, 35 and 36).

Phytophthora Tolerance: Tolerance to Phytophthora root rot is rated on ascale of 1 to 9 in the greenhouse assay, where a rating less than 3.5 isconsidered tolerant, between 3.5-6 is considered moderately tolerant,and greater than 6 indicates sensitivity to Phytophthora. (Note that ascore in the 1-2 range may indicate resistance and therefore not be atrue reflection of high tolerance to Phytophthora).

Plant Height (PHT): Plant height is taken from the top of soil to thetop node of the plant and is measured in inches.

Predicted Relative Maturity (PRM): The maturity grouping designated bythe soybean industry over a given growing area. This figure is generallydivided into tenths of a relative maturity group. Within narrowcomparisons, the difference of a tenth of a relative maturity groupequates very roughly to a day difference in maturity at harvest.

Protein (PRO), or Protein Percent: Seed protein content is measured andreported on a percentage basis.

Regeneration: The development of a plant from tissue culture.

Relative Maturity: The maturity grouping designated by the soybeanindustry over a given growing area. This figure is generally dividedinto tenths of a relative maturity group. Within narrow comparisons, thedifference of a tenth of a relative maturity group equates very roughlyto a day difference in maturity at harvest.

Seed Protein Peroxidase Activity: Seed protein peroxidase activity isdefined as a chemical taxonomic technique to separate varieties based onthe presence or absence of the peroxidase enzyme in the seed coat. Thereare two types of soybean varieties, those having high peroxidaseactivity (dark red color) and those having low peroxidase activity (nocolor).

Seed Weight (SWT): Soybean seeds vary in size; therefore, the number ofseeds required to make up one pound also varies. This affects the poundsof seed required to plant a given area, and can also impact end uses.(SW100=weight in grams of 100 seeds.)

Seed Yield (Bushels/Acre): The yield in bushels/acre is the actual yieldof the grain at harvest.

Seedling Vigor Rating (SDV): General health of the seedling, measured ona scale of 1 to 9, where 1 is best and 9 is worst.

Seeds per Pound: Soybean seeds vary in size; therefore, the number ofseeds required to make up one pound also varies. This affects the poundsof seed required to plant a given area, and can also impact end uses.

Selection Index (SELIN): The percentage of the test mean.

Self-pollination: The transfer of pollen from the anther to the stigmaof the same plant.

Shattering: The amount of pod dehiscence prior to harvest. Poddehiscence involves seeds falling from the pods to the soil. This is avisual score from 1 to 9 comparing all genotypes within a given test. Ascore of 1 means pods have not opened and no seeds have fallen out. Ascore of 5 indicates approximately 50% of the pods have opened, withseeds falling to the ground and a score of 9 indicates 100% of the podsare opened.

Single Locus Converted (Conversion) Plant: Plants which are developed bya plant breeding technique called backcrossing and/or by genetictransformation to introduce a given locus that is transgenic in origin,wherein essentially all of the morphological and physiologicalcharacteristics of a soybean variety are recovered in addition to thecharacteristics of the locus transferred into the variety via thebackcrossing technique or by genetic transformation. It is understoodthat once introduced into any soybean plant genome, a locus that istransgenic in origin (transgene), can be introduced by backcrossing aswith any other locus.

Southern Root Knot Nematode (SRKN): Greenhouse assay reaction scores arebased on severity, measured using a 1-9 scale. Resistant (R) correspondsto a rating <6.1, moderately resistant (MR) to 6.1<6.6, moderatelyresistant to moderately susceptible (MR-MS) 6.6<7.4, and susceptible (S)>7.4.

Southern Stem Canker (STC): Greenhouse assay scoring is based onpercentage of dead plants (DP). This percentage is converted to a 1-9scale: 1=no DP, 2=<10% DP, 3=10-30% DP, 4=31-40% DP, 5=41-50% DP,6=51-60% DP, 7=61-70%DP, 8=71-90% DP, 9=91-100% DP. Resistant (R)corresponds to a rating <3.9, moderately resistant (MR) 4-5.9%,moderately susceptible (MS) 6-7.9, susceptible (S) 8-8.9, and highlysusceptible (HS) >8.9.

Soybean Cyst Nematode (SCN): Greenhouse screening scores are based on afemale index % of Lee 74. Resistant (R) corresponds to a rating <10%,moderately resistant (MR) 10-21.9%, moderately resistant to moderatelysusceptible (MR-MS) 22-39.9%, and susceptible (S) >39.9%.

Stearate: A fatty acid in soybean seeds measured and reported as apercent of the total oil content.

Substantially Equivalent: A characteristic that, when compared, does notshow a statistically significant difference (e.g., p=0.05) from themean.

Sudden Death Syndrome: Leaf symptoms appear first as bright yellowchlorotic spots with progressive development of brown necrotic areas andeventual leaflet drop. Greenhouse screening plants are scored on a 1-9scale based on foliar symptom severity, measured using a 1-9 scale.Resistant (R) corresponds to a rating <3, moderately resistant (MR)3.0-4.9, moderately susceptible (MS) 5.0-6.9, susceptible (S) 7.0-8.0and highly susceptible (HS) >8.

Tissue Culture: A composition comprising isolated cells of the same or adifferent type or a collection of such cells organized into parts of aplant.

Transgene: A genetic locus comprising a sequence which has beenintroduced into the genome of a soybean plant by transformation.

Yield Best Estimate (YLD_BE): The adjusted yield of a plot inbushels/acre. Plot yields are adjusted using the nearest neighborspatial covariate method first described by Papadakis (Méthodestatistique pour des experiences sur champ, Thessaloniki Plant BreedingInstitute Bulletin No. 23, Thessaloniki, London, 1937).

Yield Count (YLD COUNT): The number of evaluated plots.

VI. Deposit Information

A deposit of the soybean variety D403301, which is disclosed hereinabove and referenced in the claims, was made with the American TypeCulture Collection (ATCC), 10801 University Blvd., Manassas, VA20110-2209. The date of deposit was Aug. 11, 2015 and the accessionnumber for those deposited seeds of soybean variety D403301 is ATCCAccession No. PTA-122415. All restrictions upon the deposit have beenremoved, and the deposit is intended to meet all of the requirements of37 C.F.R. §1.801-1.809. The deposit will be maintained in the depositoryfor a period of 30 years, or 5 years after the last request, or for theeffective life of the patent, whichever is longer, and will be replacedif necessary during that period.

* * *

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of the foregoing illustrative embodiments, itwill be apparent to those of skill in the art that variations, changes,modifications, and alterations may be applied to the composition,methods, and in the steps or in the sequence of steps of the methodsdescribed herein, without departing from the true concept, spirit, andscope of the invention. More specifically, it will be apparent thatcertain agents that are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope, and concept of the invention as defined by theappended claims.

The references cited herein, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

What is claimed is:
 1. A plant of soybean variety D403301, wherein asample of seed of said variety has been deposited under ATCC AccessionNo. PTA-122415.
 2. A plant part of the plant of claim 1, wherein theplant part comprises at least one cell of said plant.
 3. The plant partof claim 2, further defined as pollen, a meristem, a cell, or an ovule.4. A seed of soybean variety D403301, wherein a sample of seed of saidvariety has been deposited under ATCC Accession No. PTA-122415.
 5. Amethod of producing soybean seed, wherein the method comprises crossingthe plant of claim 1 with itself or a second soybean plant.
 6. Themethod of claim 5, wherein the method is further defined as comprisingcrossing the plant of soybean variety D403301with a second, distinctsoybean plant to produce an F₁ hybrid soybean seed.
 7. An F₁ hybridsoybean seed produced by the method of claim
 6. 8. An F₁ hybrid soybeanplant produced by growing the seed of claim
 7. 9. A compositioncomprising the seed of claim 4 comprised in plant seed growth media,wherein a sample of seed of said variety has been deposited under ATCCAccession No. PTA-122415.
 10. The composition of claim 9, wherein thegrowth media is soil or a synthetic cultivation medium.
 11. A plant ofsoybean variety D403301further comprising a single locus conversion,wherein a sample of seed of soybean variety D403301 has been depositedunder ATCC Accession No. PTA-122415, and wherein said plant otherwisecomprises essentially all of the morphological and physiologicalcharacteristics of soybean variety D403301 when grown under the sameenvironmental conditions.
 12. The plant of claim 11, wherein the singlelocus conversion comprises a transgene.
 13. A seed that produces theplant of claim
 11. 14. The seed of claim 13, wherein the single locusconfers a trait selected from the group consisting of male sterility,herbicide tolerance, insect resistance, pest resistance, diseaseresistance, modified fatty acid metabolism, abiotic stress resistance,altered seed amino acid composition, site-specific geneticrecombination, and modified carbohydrate metabolism.
 15. The seed ofclaim 14, wherein the single locus confers tolerance to an herbicideselected from the group consisting of glyphosate, sulfonylurea,imidazolinone, dicamba, glufosinate, phenoxy propionic acid,cyclohexanedione, triazine, benzonitrile, PPO-inhibitor herbicides, andbromoxynil.
 16. The seed of claim 13, wherein the single locusconversion comprises a transgene.
 17. The method of claim 6, wherein themethod further comprises: (a) crossing a plant grown from said F₁ hybridsoybean seed with itself or a different soybean plant to produce a seedof a progeny plant of a subsequent generation; (b) growing a progenyplant of a subsequent generation from said seed of a progeny plant of asubsequent generation and crossing the progeny plant of a subsequentgeneration with itself or a second plant to produce a progeny plant of afurther subsequent generation; and (c) repeating steps (a) and (b) usingsaid progeny plant of a further subsequent generation from step (b) inplace of the plant grown from said F₁ hybrid soybean seed in step (a),wherein steps (a) and (b) are repeated with sufficient inbreeding toproduce an inbred soybean plant derived from the soybean varietyD403301.
 18. The method of claim 17, comprising crossing said inbredsoybean plant derived from the soybean variety D403301with a plant of adifferent genotype to produce a seed of a hybrid soybean plant derivedfrom the soybean variety D403301.
 19. A method of producing a commodityplant product comprising collecting the commodity plant product from theplant of claim
 1. 20. The method of claim 19, wherein the commodityplant product is protein concentrate, protein isolate, grain, soybeanhulls, meal, flour, or oil.
 21. A soybean commodity plant productproduced by the method of claim 20, wherein the commodity plant productcomprises at least one cell of soybean variety D403301.